Transfer of an Anti-HIV-1 Ribozyme Gene into Primary Human Lymphocytes

Abstract

We reported previously that human CD4⁺ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-l viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HlV-1-infected patients in vivo.

Overview summary

In an effort to assess efficacy of an anti-HIV ribozyme in primary human cells, an RNA polymerase III driven, anti-US hairpin ribozyme gene was stably introduced into human peripheral blood lymphocytes via an amphotropic retroviral vector. Transduced cells were selected by resistance to G418 and subsequently challenged with HXB2 or HIV-1 clinical isolates. Ribozyme expressing cultures showed up to 100-fold reduction in virus production as compared to LNL-6-transduced controls. Such inhibition was donor independent and persistent, demonstrating that ex vivo transduction of a ribozyme gene can be utilized to produce HIV-1-resistant peripheral blood lymphocytes.